FIGURE 5.

IL-2-mediated tyrosine phosphorylation of IL-2Rβ is inhibited by CA pretreatment in primary human lymphocytes and YT cells. A, quiescent PHA-activated normal human PBMCs were left untreated (lane a), treated with 100 nm CA for 60 min (lane b), stimulated with IL-2 for 10 min (lane c), or pretreated with 100 nm CA for 90 min prior to stimulation with IL-2 for 10 min (lane d). The cells were lysed, the IL-2Rβ was immunoprecipitated (IP) and analyzed by Western blot (WB) using the indicated antibodies. B and C, YT cells (1.5 × 10⁷) were labeled with [³²P]orthophosphate for 2 h at 37 °C and treated without (lanes a and c) or with 100 nm CA (lanes b and d) for 60 min prior to stimulation in the absence (lanes a and b) or presence (lanes c and d) of IL-2 for 10 min. IL-2Rβ was immunoprecipitated from cell lysates, separated by SDS-PAGE, and subjected to autoradiography (left panel). Phosphoamino acid analysis was performed on the corresponding IL-2Rβ bands (lanes a–d) (right panel). The position of phosphoserine, -threonine, and -tyrosine standards (pS, pT, and pY) were detected by ninhydrin as indicated (circles). C, YT cells were left untreated (lane a), treated with 100 nm CA for 60 min (lane b), stimulated with 100 nm IL-2 for 10 min (lane c), or pretreated with 100 nm CA for 10–150 min followed by stimulation with IL-2 for 10 min (lanes d–i). IL-2Rβ IPs were assessed by Western blot analysis using the indicated antibodies. Representative data from three independent experiments are shown.